BIDMC Mass Spectrometry Facility In-Gel Digestion Protocol
1. Staining: Coomassie Brilliant Blue stain – any standard CBB staining protocol;
keep staining time relatively short, thoroughly destain
2. Storage: CBB washed with 50% acetonitrile and stored damp. After excision of
bands, put in 1.5mL plain microfuge tube and wash with 150-200 L of 50%
acetonitrile for 15 min and discard supernatant. Store in same tube, moist, not
submerged, frozen at –20 or below.
3. Gel Band Preparation: The gel pieces are cut in 1mm x 1mm cubes and washed
with 150-200 L of 50% acetonitrile for 15 min and the gel pieces are then dried
in a SpeedVac.
4. Reduction: Reduce gel with a volume sufficient to cover the gel pieces (~10µL),
of 10mM DTT in 100mM NH4HCO3 at 56C for 30 min.
5. Alkylation: After cooled to RT, the DTT is removed, add 3-4 times the gel
volume of acetonitrile to shrink the gel pieces and replace with the same volume
of 55mM iodoacetamide in 100mM NH4HCO3 that was used for reduction and
keep in the dark (I use a drawer), RT, for 20 min.
6. Washes: Wash gel pieces with 150µL 100mM NH4HCO3 and incubate for 10
min. Discard buffer and shrink the gel piece with 50L acetonitrile (remove all
liquid), swell with 50L 100mM NH4HCO3 again and dehydrate/shrink with the
same volume of acetonitrile.
7. Drying: Remove all liquid and completely dry gel in SpeedVac for ~15 min.
8. Rehydration with Digestion Buffer and Addition of Trypsin: Rehydrate the
gel pieces in 10-20µL (depending on the gel size) of 100mM NH4HCO3 and
25ng/µL of trypsin (195L 50mM NH4HCO3 + 5L of 1g/L trypsin in 50mM
acetic acid [stock soln.]) and wait 15 min. on ice. After 15 min., check and add
more trypsin buffer if necessary. Add an additional 10-20 L of 50mM NH4HCO3
(no trypsin).
9. Digestion: Digest overnight at 37oC in thermomixer.
10. Peptide Extraction and Pooling: Add 25L 20mM NH4HCO3 (depending on the
gel size) and incubate for 10 min at 37oC with shaking. Spin down and collect
supernatant and add to a plain 1.5mL microfuge tube. Add 50L of 5% formic
acid/40% acetonitrile and incubate for 15 min. at 37 oC with shaking. Spin down
and add supernatant to first extraction.
11. Final Volume: Cautiously dry down on SpeedVac to discussed volume. Not to
dryness. In general: < 5pmoles dry to 10µL, up to ~50 pmol dry to 20µL, > 50
pmol dry to 50µL. The volume is then transferred to a FAMOS autosampler vial
for LC/MS/MS or spotted onto a MALDI plate after LC or ZipTip C18 clean-up.